CN111534456A - Composite lactic acid bacteria agent and application thereof in silage of sugarcane tail leaves - Google Patents
Composite lactic acid bacteria agent and application thereof in silage of sugarcane tail leaves Download PDFInfo
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- CN111534456A CN111534456A CN202010258231.8A CN202010258231A CN111534456A CN 111534456 A CN111534456 A CN 111534456A CN 202010258231 A CN202010258231 A CN 202010258231A CN 111534456 A CN111534456 A CN 111534456A
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- silage
- sugarcane
- lactobacillus
- lactobacillus plantarum
- aerobic
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- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 20
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
- A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
- A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
- A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
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- A—HUMAN NECESSITIES
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- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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Abstract
The invention discloses a composite lactic acid bacteria agent and application thereof in silage of sugarcane tail leaves. The composite Lactobacillus for the silage of the sugarcane tail leaves comprises the active ingredients of Lactobacillus plantarum WQ-01 and Lactobacillus hilgardii60TS-2, and can maintain aerobic stability, pH and temperature of the silage of the sugarcane tail leaves, maintain the relative abundance of the Lactobacillus in microbial flora and reduce the dry matter loss after aerobic exposure. The composite lactic acid bacteria agent comprises two types of lactic acid bacteria of homotype fermentation and heterotype fermentation, can promote the rapid fermentation of the sugarcane tail leaves in the initial stage of ensiling, and can prolong the aerobic stabilization time after contacting air, thereby improving the nutritive value of the sugarcane tail leaf ensiling and prolonging the storage life of the sugarcane tail leaf ensiling.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a compound microbial inoculant and application thereof, in particular to a compound lactic acid inoculant and application thereof in silage of sugarcane tail leaves.
Background
Sugarcane belongs to C-C4 plants and has high photosynthetic utilization rate. The planting area of sugarcane in China is the third in the world, the total annual sugarcane yield is 1.17 hundred million tons, the tail leaves of the sugarcane account for 20 percent of the fresh weight of the whole sugarcane plant, and the annual sugarcane tip yield in China is 2300 million tons. The conventional method for treating the sugarcane tail leaves is to discard or burn, which not only causes resource waste but also pollutes the environment. The tail leaves of the sugarcane can be used as coarse fodder for cattle and sheep in winter and spring, the storage time can be prolonged through silage, the palatability of the tail leaves of the sugarcane is improved, and therefore the feeding efficiency is improved.
In subtropical areas of Guangxi, the perennial rainy period, especially the 11 to 4 months of the year after the harvest of the tail leaves of the sugarcane, the high-moisture tail leaves of the sugarcane are difficult to be ensiled, and the number of background yeast bacteria is up to 105The fresh weight of CFU/g easily causes nutrition loss in the initial fermentation stage, the mould is easily bred in a high-humidity environment, secondary fermentation is easily generated after aerobic exposure, and the mildewing loss is very serious.
Disclosure of Invention
The invention aims to provide a composite lactic acid bacteria agent and application thereof in silage of sugarcane tail leaves.
In a first aspect, the present invention protects a new use of Lactobacillus plantarum and Lactobacillus hilgardii.
The invention protects the application of Lactobacillus plantarum and Lactobacillus hilgardii in preparing sugarcane silage or sugarcane silage additives.
In a second aspect, the invention features a bacterial agent.
The active ingredients of the microbial inoculum protected by the invention are Lactobacillus plantarum and Lactobacillus hilgardii.
Further, the microbial inoculum at least contains 1.5 × 10 per milliliter7The lactobacillus plantarum strain is characterized in that each milliliter of the strain contains at least 1.5 × 107Lactobacillus hilgardii, 1.5 × 10/ml of the inoculum in a specific example of the invention7CFU plant breast rodThe bacterial strain Lactobacillus plantarum contains 1.5 × 10 per ml7Lactobacillus hilgardii, CFU.
Further, the ratio of CFU of Lactobacillus plantarum to CFU of Lactobacillus hilgardii is 1: (1-10). In a specific embodiment of the invention, the ratio of CFU of the Lactobacillus plantarum and the Lactobacillus hilgardii is 1: 1.
in a third aspect, the invention protects a preparation method of the microbial inoculum.
The preparation method of the microbial inoculum protected by the invention comprises the step of uniformly mixing the Lactobacillus plantarum microbial inoculum and the Lactobacillus hilgardii microbial inoculum with a solvent.
Further, the preparation method of the Lactobacillus plantarum microbial inoculum comprises the following steps: inoculating Lactobacillus plantarum in culture medium, culturing, centrifuging, collecting thallus precipitate, and diluting to OD 6001, obtaining Lactobacillus plantarum bacterial agent.
The preparation method of the Lactobacillus hilgardii microbial inoculum comprises the following steps: inoculating Lactobacillus hilgardii into culture medium, culturing, centrifuging, collecting thallus precipitate, and diluting to OD 6001, Lactobacillus hilgardii inoculum was obtained.
Further, the culture medium may specifically be an MRS liquid culture medium.
The culture condition can be specifically static culture at 37 ℃ for 48 h.
The centrifugation condition can be specifically centrifugation at 8500rpm for 10min at 4 ℃.
Diluting the bacterial pellet to OD with sterile physiological saline 6001. The formula of the sterile normal saline is as follows: 8.5g of sodium chloride was dissolved in 1L of sterile water to obtain the sterile physiological saline.
Lactobacillus plantarum p in Lactobacillus plantarum microbial inoculumThe lantarum concentration is specifically 1 × 109CFU/mL。
The concentration of Lactobacillus hilgardii in the Lactobacillus hilgardii microbial inoculum is specifically 5 × 108CFU/mL。
The solvent may specifically be sterile physiological saline.
The volume ratio of the Lactobacillus plantarum microbial inoculum to the Lactobacillus hilgardii microbial inoculum can be specifically 1: 2.
In a specific embodiment of the invention, 0.75mL of Lactobacillus plantarum microbial inoculum, 1.5mL of Lactobacillus hilgardii microbial inoculum and sterile normal saline are mixed uniformly until the total volume is 50mL, so as to obtain the microbial inoculum, namely the composite lactic acid microbial inoculum of the invention.
In a fourth aspect, the present invention protects a new use of the above microbial inoculum or a microbial inoculum prepared according to the above method.
The invention protects the application of the microbial inoculum or the microbial inoculum prepared by the method in any one of the following A1) -A10):
A1) preparing sugarcane silage;
A2) as an additive to sugar cane silage;
A3) reducing the loss of dry matter in the sugarcane silage;
A4) the stability of the content of organic acid (such as lactic acid and acetic acid) in the sugarcane silage is maintained;
A5) inhibiting the increase of the content of ammoniacal nitrogen in the sugarcane silage;
A6) maintaining the aerobic stability of the sugarcane silage;
A7) maintaining the abundance of lactobacillus in the sugarcane silage;
A8) inhibiting the proliferation of aerobic spoilage bacteria (such as Acetobacter) in sugarcane silage;
A9) the nutritive value of the sugarcane silage is improved;
A10) the storage life of the sugarcane silage is prolonged.
In a fourth aspect, the invention provides a preparation method of sugarcane silage.
The preparation method of the sugarcane silage protected by the invention comprises the following steps:
the method A comprises the following steps: and (3) applying the microbial inoculum or the microbial inoculum prepared by the method to sugarcane for silage to obtain sugarcane silage.
The method B comprises the following steps: the Lactobacillus plantarum and Lactobacillus hilgardii are applied to sugar cane for silage, and sugar cane silage is obtained.
Further, in the method A, the application amount of the microbial inoculum is 50 mL/1.5 kg (fresh weight of sugarcane).
In the method B, the application amount of the Lactobacillus plantarum and the Lactobacillus hilgardii is 5 × 105CFU/g (fresh weight of sugarcane).
Further, the ensiling condition is that the ensiling is carried out for 90 days at 15-25 ℃ under the condition of keeping out the light.
In any of the above-described microbial agents or methods or uses, the Lactobacillus plantarum is Lactobacillus plantarum WQ-01;
the Lactobacillus hilgardii is Lactobacillus hilgardii60 TS-2.
The sugarcane is sugarcane tail leaves.
The use of the above-described method for reducing the loss of dry matter in sugarcane silage and/or for maintaining the stability of the organic acid content in sugarcane silage and/or for inhibiting the increase of the ammoniacal nitrogen content in sugarcane silage and/or for maintaining the aerobic stability of sugarcane silage and/or for maintaining the abundance of lactobacillus in sugarcane silage and/or for inhibiting the proliferation of aerobic spoilage bacteria in sugarcane silage and/or for increasing the nutritional value of sugarcane silage and/or for prolonging the shelf life of sugarcane silage also belongs to the scope of the invention.
In a fifth aspect, the invention protects Lactobacillus plantarum WQ-01.
The preservation number of the Lactobacillus plantarum WQ-01 protected by the invention is CGMCC No. 13318. The strain has been preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of West Lu 1 of Beijing, Tokyo, sunny district, Ministry of China microbiology, postal code 100101) at 2016, 11/18/2016, with the preservation number of CGMCC No. 13318.
In a sixth aspect, the invention provides a Lactobacillus hilgardii60 TS-2.
The preservation number of the Lactobacillus hilgardii60TS-2 protected by the invention is CGMCC No. 19435. The strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address is No. 3 of the institute of microbiology of the national academy of sciences of China, No. 100101 of No.1 of the West Lu of the North Kyoho, Beijing city) within 3 and 4 days of 2020, and the preservation number is CGMCC No. 19435.
The invention aims to provide a composite Lactobacillus for silage of sugarcane tail leaves, which comprises Lactobacillus plantarum (Lactobacillus plantarum) WQ-01 and Lactobacillus hilgardii (Lactobacillus hilgardii)60TS-2, can maintain aerobic stability, pH and temperature of the silage of the sugarcane tail leaves, keeps relative abundance of Lactobacillus in microbial flora, and reduces dry matter loss after aerobic exposure. The composite lactic acid bacteria agent comprises two types of lactic acid bacteria of homotype fermentation and heterotype fermentation, can promote the rapid fermentation of the sugarcane tail leaves in the initial stage of ensiling, and can prolong the aerobic stabilization time after contacting air, thereby improving the nutritive value of the sugarcane tail leaf ensiling and prolonging the storage life of the sugarcane tail leaf ensiling.
Deposit description
The strain name is as follows: lactobacillus plantarum
Latin name: lactobacillus plantarum
The strain number is as follows: WQ-01
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 2016 (11 months) and 18 days
Registration number of the preservation center: CGMCC No.13318
The strain name is as follows: lactobacillus hilgardii
Latin name: lactobacillus hilgardii
The strain number is as follows: 60TS-2
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 3/month/4/2020
Registration number of the preservation center: CGMCC No.19435
Drawings
FIG. 1 is a graph showing the effect of a complex lactobacillus on the dry matter content of sugarcane tail leaf silage after oxygen exposure.
FIG. 2 is a graph showing the effect of a complex lactobacillus on dry matter loss after oxygen exposure of sugarcane tail leaf silage.
FIG. 3 shows the effect of the complex lactobacillus on pH after oxygen exposure of sugarcane tail leaf silage.
FIG. 4 shows the effect of the composite lactobacillus on the content of lactic acid after the silage of sugarcane tail leaves is exposed to oxygen.
FIG. 5 is a graph showing the effect of a composite lactobacillus on the acetic acid content of sugarcane tail leaf silage after oxygen exposure.
FIG. 6 shows the effect of the composite lactobacillus on the ammoniacal nitrogen content of sugarcane tail leaf silage after oxygen exposure.
FIG. 7 shows the effect of the complex lactobacillus on the temperature of sugarcane tail leaf after oxygen exposure.
FIG. 8 shows the effect of the complex lactobacillus on the microbial flora of sugarcane tail leaf after aerobic exposure.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 isolation, identification and preservation of strains
First, isolation of the Strain
Lactobacillus plantarum WQ-01 was isolated from sweet sorghum and Lactobacillus hilgardii60TS-2 from sugarcane tail leaves. The specific separation method is as follows: taking 10g of sweet sorghum or sugarcane tail leaves, adding 90mL of normal saline, and sequentially diluting the mixture to 10-7Selecting appropriate gradient coating on MRS culture medium (1L MRS culture medium formula is 10g of beef extract, 10g of casein peptone, 5g of yeast extract, 20g of glucose, 5g of sodium acetate, 2g of citric acid hydrogen diamine, Tween 801 mL, K)2HPO42g，MgSO4·7H2O 0.58g，MnSO4·7H2O0.25g, agar powder 1.5%, double distilled water to lL, and autoclaving). And (3) performing static culture at 37 ℃ for 48h, selecting the monoclonal antibody with smooth edges, transferring the monoclonal antibody to an MRS liquid culture medium, continuing the static culture at 37 ℃ for 48h, and storing the culture solution into a freezing storage tube with the final concentration of 25% glycerol.
II, molecular identification of strains
The 16S rDNA sequences of the WQ-01 strain and the 60TS-2 strain were amplified and sequenced. The primer sequences are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3' are provided.
The 16S rDNA sequence of the WQ-01 strain is shown as a sequence 1 in a sequence table. The 16S rDNA sequence of the 60TS-2 strain is shown as a sequence 2 in a sequence table. The similarity of the 16S rDNA sequence of the WQ-01 strain to Lactobacillus plantarum in the NCBI database was 99.79%, and the similarity of the 16S rDNA sequence of the 60TS-2 strain to Lactobacillus hilgardii in the NCBI database was 99.67%.
Third, preservation of the Strain
By combining the above identification results, the WQ-01 strain is determined to be Lactobacillus plantarum in name, which is classified and named Lactobacillus plantarum, and the strain is preserved in the China general microbiological culture Collection center (CGMCC for short, address: No. 3 of Beijing university North Chen Xilu 1 institute of Inward Yangtze, China academy of sciences, postal code 100101) in 2016, 11/18 days, and the preservation number is CGMCC No. 13318.
By combining the above identification results, it was determined that the 60TS-2 strain named Lactobacillus hilgardii, which was deposited in China general microbiological culture Collection center (CGMCC for short, address: Beijing, West Luo No.1 Hokko 3, institute of microbiology, Japan academy of sciences, postal code 100101) at 3.4.2020, and the collection number is CGMCC No. 19435.
Fourthly, biological characteristics of the strain
Lactobacillus plantarum WQ-01 and Lactobacillus hilgardii60TS-2 were grown in MRS liquid medium (Beijing Luqiao technology, Inc., product number CM187) or MRS solid medium (1.5% agar powder was added to MRS liquid medium), and anaerobically cultured at 37 ℃. The results of the biological characterization are shown in Table 1.
The results show that: the Lactobacillus plantarum WQ-01 and Lactobacillus hilgardii60TS-2 are gram-positive bacteria, rod-shaped structures, catalase-negative, oxidase-negative, and tolerant to NaCl concentrations of 3.0% and 6.5%. The lactobacillus plantarum L.plantarum WQ-01 is a homotypic fermentation strain, can grow within the pH range of 3.5-8.5, has weaker growth at the pH of 2.0, and grows at the temperature of 15-45 ℃; lactobacillus hilgardii60TS-2 is a heterotypic fermentation strain that can grow at pH 3.5-8.0, weakly at pH2.0, at 15-40 ℃, and weakly at 45 ℃.
TABLE 1 Strain characterization of Lactobacillus plantarum WQ-01 and Lactobacillus hilgardii60TS-2
| Characteristics of | L.plantarum WQ-01 | L.hilgardii 60TS-2 |
| Storage weaving machineNumber (C) | CGMCC No.13318 | CGMCC No.19435 |
| Shape of | Rod-like structure | Rod-like structure |
| Gram stain | + | + |
| Type of fermentation | Homofermentation homofermentations | Heterofermentation Heterofermentation |
| Catalase enzyme | - | - |
| Oxidase enzyme | - | - |
| Growth pH | ||
| 2.0 | W | W |
| 3.5 | + | + |
| 4.0 | + | + |
| 4.5 | + | + |
| 8.0 | + | + |
| 8.5 | + | - |
| 9.0 | - | - |
| Tolerant to NaCl | ||
| 3.0％ | + | + |
| 6.5％ | + | + |
| |
||
| 10℃ | - | - |
| 15℃ | + | + |
| 20℃ | + | + |
| 25℃ | + | + |
| 30℃ | + | + |
| 35℃ | + | + |
| 40℃ | + | + |
| 45℃ | + | W |
Note: + is positive, -negative, W is weakly positive.
Example 2 preparation and application of composite lactic acid bacteria agent
Preparation of composite lactic acid bacteria
1. Preparation of lactobacillus plantarum WQ-01 microbial inoculum
Will plantInoculating Lactobacillus WQ-01 in MRS liquid culture medium, standing at 37 deg.C for 48 hr, centrifuging at 4 deg.C at 8500rpm for 10min, collecting thallus precipitate, and diluting with sterile physiological saline to OD 6001, obtaining a lactobacillus plantarum WQ-01 bacterial preparation (liquid), wherein the concentration of the lactobacillus plantarum WQ-01 in the lactobacillus plantarum WQ-01 bacterial preparation (liquid) is 1 × 109CFU/mL。
2. Preparation of lactobacillus hilgardii60TS-2 microbial inoculum
Inoculating Lactobacillus hilgardii60TS-2 into MRS liquid culture medium, standing at 37 deg.C for 48 hr, centrifuging at 4 deg.C at 8500rpm for 10min, collecting thallus precipitate, and diluting the thallus precipitate with sterile physiological saline to OD600Obtaining the lactobacillus hilgardii60TS-2 microbial inoculum (liquid), wherein the concentration of the lactobacillus hilgardii60TS-2 in the lactobacillus hilgardii60TS-2 microbial inoculum (liquid) is 5 × 108CFU/mL。
3. Composite lactic acid bacteria agent
0.75mL of lactobacillus plantarum WQ-01 microbial inoculum, 1.5mL of lactobacillus hilgardii60TS-2 microbial inoculum and sterile normal saline are mixed uniformly until the total volume is 50mL to obtain the composite lactic acid bacterial inoculum, wherein each 50mL of the composite lactic acid bacterial inoculum contains 0.75 × 109CFU Lactobacillus plantarum WQ-01 and 0.75 × 109CFU Lactobacillus hilgardii60 TS-2.
Second, ensiling of sugarcane
1. Test materials: cutting the tail leaves of fresh sugarcane from a test field of Guangxi Zhuang autonomous region livestock research institute until the length of the tail leaves is 2-4 cm.
2. Test method
Control group (CK): 50mL of physiological saline was sprayed on 1.5kg of fresh sugarcane tail leaves.
Test group (LHLP) 50mL of the complex lactic acid bacteria were all sprayed on 1.5kg of fresh sugarcane tail leaves, and the final concentrations of Lactobacillus plantarum WQ-01 and Lactobacillus hilgardii60TS-2 were both 5 × 105CFU/g fresh weight of tail leaves of sugarcane.
And (3) respectively ensiling the sugarcane tail leaves treated by the test group and the control group into polyethylene bags, repeating the treatment for 3 times, vacuumizing, and storing for 90 days at 15-25 ℃ in the dark to obtain the sugarcane tail leaf ensiling feed. The bags were opened after 90 days of storage (aerobic exposure).
Third, detection of silage effect of sugarcane
1. Dry Matter (DM) detection
In order to study the influence of the compound lactobacillus on dry matters in the silage of sugarcane tail leaves, the dry matter content of a silage sugarcane sample is detected. The method comprises the following specific steps:
the bags are opened after 90 days of storage, and samples are collected for dry matter detection at 0, 1, 2, 3, 4 and 5 days after opening the bags respectively. The dry matter detection steps are as follows: weighing about 100g of sample, drying the sample in a constant temperature dryer at 65 ℃ to constant weight, naturally cooling the sample to room temperature, weighing the sample, and calculating the content of dry substances in the sample. Aerobic exposure 5 days dry matter loss-aerobic exposure 0 days dry matter content-aerobic dew 5 days dry matter content.
Results as shown in fig. 1 and 2, after 90 days of ensiling, the dry matter content (26.11%) of the control group (CK) was higher than that (25.35%) of the test group (LHLP), but after 5 days of aerobic exposure, the dry matter content (24.76%) of the LHLP group was higher than that (24.42%) of the control group. The LHLP group dry matter loss was significantly lower (0.59%) than the CK group (1.69%) after 5 days of aerobic exposure. The composite lactic acid bacteria agent can better preserve the dry matter content in the sugarcane tail leaf silage and reduce the dry matter loss in the sugarcane silage.
2. Fermentation product detection
In order to study the effect of the compound lactobacillus on the fermentation product in the silage of the sugarcane tail leaves, the pH, organic acids (lactic acid and acetic acid) and ammonia nitrogen of the silage sugarcane samples are detected. The method comprises the following specific steps:
the bags were opened after 90 days of storage, and samples were taken for pH and organic acid detection at 0, 1, 2, 3, 4, and 5 days after opening the bags, respectively. 25g of silage sugarcane tail leaves are taken, 225mL of normal saline is added, and the mixture is uniformly mixed and placed in a refrigerator at 4 ℃ for overnight placement. The pH, organic acid and ammonia nitrogen were then measured in a 50mL centrifuge tube using a 4-layer gauze filter. The pH value is determined using a pH meter (pH 213; HANNA; Italy). The organic acid content was determined by HPLC (1200, Agilent, America) equipped with a UV detector (210nm) and a column (ICSep COREGEL-87H), with a mobile phase of 0.005M H at 55 ℃2SO4The flow rate was 0.6 mL/min. The ammoniacal nitrogen content was determined using phenol hypochlorite and ninhydrin colorimetry.
As shown in fig. 3, the pH of the LHLP group was slightly higher than that of the CK group (pH 3.71) after 90 days of ensiling, and was 3.82. But after 2 days of aerobic exposure, the CK group pH increased dramatically until 4.95 after 5 days of aerobic exposure; the pH of the LHLP group was nearly stable with a pH of below 4.0 throughout 5 days of aerobic exposure.
As shown in FIG. 4, the lactic acid content of the LHLP group was lower after 90 days of ensiling, 66.26g/kg DM compared to the CK group (107.43g/kg DM), but the lactic acid content of the CK group gradually decreased after aerobic exposure until 31.6g/kg DM after 5 days of aerobic exposure, while the lactic acid content of the LHLP group remained relatively stable and remained at 65g/kg DM or more after 5 days of aerobic exposure.
As shown in FIG. 5, after 90 days of ensiling, the acetic acid content of the LHLP group was higher, 38.68g/kg DM, compared to the CK group (15.66g/kg DM), and gradually decreased after aerobic exposure until 2.88g/kg DM after 5 days of aerobic exposure, while the acetic acid content of the LHLP group remained at a more stable level, with the acetic acid content of 26.75g/kg DM after 5 days of aerobic exposure being 9 times that of the CK group.
As shown in FIG. 6, the ammonia nitrogen content of both CK group and LHLP group was less than 1g/kg DM after 90 days of ensiling, whereas after 3 days of aerobic exposure, the ammonia nitrogen content of CK group was gradually increased to 1.74g/kg DM, which was 2 times the initial value of 0.83g/kg DM, and the ammonia nitrogen content of LHLP group was almost unchanged after aerobic exposure, and remained at a low level of 0.68g/kg DM at 5 days of aerobic exposure.
The results show that the compound lactobacillus can maintain the stability of the content of lactic acid and acetic acid in the silage of sugarcane tail leaves and inhibit the increase of the content of ammoniacal nitrogen.
3. Aerobic stability assay
In order to study the influence of the compound lactobacillus on the aerobic stability of the silage of the sugarcane tail leaves, the aerobic stability of the silage sugarcane sample is detected. The method comprises the following specific steps:
after 90 days of ensiling, the ensiling bag was opened, and the temperature was measured by a multichannel data recorder (SMOWO, model: MDL-1048A; Shanghai Tianhe Automation instruments Co., Ltd., China) set to record every 4 hours. To prevent drying and contamination, the sample bag was covered with a double layer of gauze. Aerobic spoilage is defined when the silage temperature rises above 2 ℃ above ambient.
As shown in FIG. 7, the temperature of CK group after 20h of aerobic exposure was 28.5 ℃ and 2 ℃ higher than room temperature (26.5 ℃), which resulted in aerobic decay, and the temperature of LHLP group after 64h of aerobic exposure reached 28.5 ℃ and the aerobic stability time was 3 times that of CK group. Furthermore, the maximum temperature of the CK group was 32.3 ℃ (after aerobic exposure for 40.3 h), and the maximum temperature of the LHLP group was only 29.2 ℃ (after aerobic exposure for 68.3 h), which was lower than that of the CK group. The composite lactic acid bacteria agent is helpful for maintaining the aerobic stability of the silage of the sugarcane tail leaves, thereby maintaining the quality of the silage after the sugarcane tail leaves are bagged.
4. Microbial flora detection
In order to research the influence of the compound lactobacillus on the microbial flora in the silage of the sugarcane tail leaves, the microbial flora of the silage sugarcane sample is detected. The method comprises the following specific steps:
after ensiling for 90 days, opening the ensiling bag to expose the ensiling bag in an aerobic way, taking 10g of ensiling samples on the 0 th day and the 2 nd day after the aerobic exposure, adding 90mL of sterile normal saline, oscillating for 30min, filtering the solution by 4 layers of gauze, and filtering the solution into a 50mL sterile centrifuge tube; centrifuging at 4 deg.C at 10000rpm for 15min, and removing supernatant; the precipitate was blown up with 1mL of sterile physiological saline, transferred to a 1.5mL centrifuge tube, centrifuged at 10000rpm at 4 ℃ for 15min, the supernatant was discarded, and the precipitate was collected. The DNA of the microorganism was extracted using the kit (MP, cat No. 12888-100). High-throughput sequencing of the extracted DNA was performed and microbial flora abundance was calculated. The abundance of the microbial flora was calculated as follows: after high-throughput sequencing, splicing the obtained sequencing sequences, obtaining clean high-quality sequences after quality control, and then flattening all samples, wherein the abundance of the microorganisms of a certain species is the proportion of the sequence number of the species to the total sequence number of the microorganisms of the samples.
As a result, as shown in fig. 8, after 90 days of ensiling, the abundance of Lactobacillus (Lactobacillus) in the LHLP group was higher than that of CK group (76.02%), accounting for 99.81% of the abundance of the microbial flora, while the relative abundance of Acetobacter (Acetobacter) was lower; after 2 days of aerobic exposure, i.e. the CK group had suffered aerobic spoilage, the relative abundance of the CK group lactobacillus decreased to 6.23%, which was much lower than that of the LHLP group lactobacillus (73.54%), whereas the relative abundance of the acetobacter increased to 93.66%, which was much higher than that of the LHLP group acetobacter (26.39%). Lactobacillus dominates ensiling with good quality of ensiling, heterotypic fermented lactic acid bacteria in lactobacillus contribute to aerobic stabilization of ensiling, and acetobacter is precisely the key microorganism responsible for aerobic spoilage of ensiling. The composite lactic acid bacteria agent is helpful for maintaining the abundance of lactobacillus in the silage of the sugarcane tail leaves and inhibiting the proliferation of aerobic putrefying bacteria.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> institute of microbiology of Chinese academy of sciences
<120> composite lactic acid bacteria agent and application thereof in silage of sugarcane tail leaves
<160>2
<170>PatentIn version 3.5
<210>1
<211>1427
<212>DNA
<213>Artificial Sequence
<400>1
caagtcgaac gaactctggt attgattggt gcttgcatca tgatttacat ttgagtgagt 60
ggcgaactgg tgagtaacac gtgggaaacc tgcccagaag cgggggataa cacctggaaa 120
cagatgctaa tatttcataa caacttggac cgcatggtcc gagtttgaaa gatggcttcg 180
gctatcactt ttggatggtc ccgcggcgta ttagctagat ggtggggtaa cggctcacca 240
tggcaatgat acgtagccga cctgagaggg taatcggcca cattgggact gagacacggc 300
ccaaactcct acgggaggca gcagtaggga atcttccaca atggacgaaa gtctgatgga 360
gcaacgccgc gtgagtgaag aagggtttcg gctcgtaaaa ctctgttgtt aaagaagaac 420
atatctgaga gtaactgttc aggtattgac ggtatttaac cagaaagcca cggctaacta 480
cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggattta ttgggcgtaa 540
agcgagcgca ggcggttttt taagtctgat gtgaaagcct tcggctcaac cgaagaagtg 600
catcggaaac tgggaaactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg 660
aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgtaactga 720
cgctgaggct cgaaagtatg ggtagcaaac aggattagat accctggtag tccataccgt 780
aaacgatgaa tgctaagtgt tggagggttt ccgcccttca gtgctgcagc taacgcatta 840
agcattccgc ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc 900
gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaagaacctt accaggtctt 960
gacatactat gcaaatctaa gagattagac gttcccttcg gggacatgga tacaggtggt 1020
gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080
ccttattatc agttgccagc attaagttgg gcactctggt gagactgccg gtgacaaacc 1140
ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200
ctacaatgga tggtacaacg agttgcgaac tcgcgagagt aagctaatct cttaaagcca 1260
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<210>2
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gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac gcgtcttggt caatgaagtt 60
gagtgcttgc atttaactga tccacattaa gacgagtggc gaactggtga gtaacacgtg 120
ggtaacttgc cccgaagcgg gggataacat ttggaaacag gtgctaatac cgcataacaa 180
caaaaaccac atggtttttg tttaaaagat ggtttcggct atcactttgg gatggacccg 240
cggcgtatta gctagttggt gaggtaatgg ctcaccaagg caatgatacg tagccgacct 300
gagagggtaa tcggccacat tgggactgag acacggccca aactcctacg ggaggcagca 360
gtagggaatc ttccacaatg gacgaaagtc tgatggagca acgccgcgtg agtgatgaag 420
ggtttcggct cgtaaaactc tgttgttgga gaagaacggg tgtcagagta actgttgaca 480
tcgtgacggt atccaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac 540
gtaggtggca agcgttgtcc ggatttattg ggcgtaaagc gagcgcaggc ggttttttag 600
gtctgatgtg aaagccttcg gcttaaccgg agaagtgcat cggaaaccgg gagacttgag 660
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caccagtggc gaaggcggct gtctggtctg caactgacgc tgaggctcga aagcatgggt 780
agcgaacagg attagatacc ctggtagtcc atgccgtaaa cgatgagtgc taagtgttgg 840
agggtttccg cccttcagtg ctgcagctaa cgcattaagc actccgcctg gggagtacga 900
ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt 960
ttaattcgat gctacgcgaa gaaccttacc aggtcttgac atcttctgct aacctaagag 1020
attaggcgtt cccttcgggg acggaatgac aggtggtgca tggttgtcgt cagctcgtgt 1080
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tagttgggca ctctggcgag actgccggtg acaaaccgga ggaaggtggg gatgacgtca 1200
aatcatcatg ccccttatga cctgggctac acacgtgcta caatggacgg tacaacgagt 1260
cgcgaaaccg cgaggtcaag ctaatctctt aaagccgttc tcagttcgga ttgtaggctg 1320
caactcgcct acatgaagtt ggaatcgcta gtaatcgtgg atcagcatgc cacggtgaat 1380
acgttcttgg gccttgtaca caccgcccgt cacaccatga gagtttgtaa cacccaaagc 1440
cggtgaggta accttcgggg gccagccgtc taaggtggga cagatgatta gggtg 1495
Claims (10)
1. Use of Lactobacillus plantarum and Lactobacillus hilgardii for the preparation of sugarcane silage or sugarcane silage additive.
2. A microbial preparation contains Lactobacillus plantarum and Lactobacillus hilgardii as active ingredients.
3. The microbial preparation according to claim 2, wherein the microbial preparation contains at least 1.5 × 10/ml7The Lactobacillus plantarum strain is characterized in that each milliliter of the strain contains at least 1.5 × 107Lactobacillus hilgardii, CFU.
4. The microbial inoculum according to claim 2 or 3, characterized in that: the ratio of the CFU of the Lactobacillus plantarum and the Lactobacillus hilgardii is 1: (1-10).
5. A method for producing the microbial agent according to any one of claims 2 to 4, comprising the step of mixing a Lactobacillus plantarum microbial agent and a Lactobacillus hilgardii microbial agent with a solvent.
6. Use of the bacterial agent of any one of claims 2 to 4 or prepared according to the process of claim 5 in any one of the following A1) -A10):
A1) preparing sugarcane silage;
A2) as an additive to sugar cane silage;
A3) reducing the loss of dry matter in the sugarcane silage;
A4) the stability of the content of organic acid in the sugarcane silage is maintained;
A5) inhibiting the increase of the content of ammoniacal nitrogen in the sugarcane silage;
A6) maintaining the aerobic stability of the sugarcane silage;
A7) maintaining the abundance of lactobacillus in the sugarcane silage;
A8) inhibiting the proliferation of aerobic spoilage bacteria in the sugarcane silage;
A9) the nutritive value of the sugarcane silage is improved;
A10) the storage life of the sugarcane silage is prolonged.
7. A preparation method of sugarcane silage comprises the following steps: applying the microbial inoculum according to any one of claims 2 to 4 or prepared according to the method of claim 5 to sugarcane for ensiling to obtain sugarcane silage;
or, the preparation method of the sugarcane silage comprises the following steps: the Lactobacillus plantarum and Lactobacillus hilgardii are applied to sugarcane for silage, and sugarcane silage is obtained.
8. Use according to claim 1 or an agent according to any one of claims 2 to 4 or a method according to claim 5 or 7 or a use according to claim 6, wherein: the Lactobacillus plantarum is Lactobacillus plantarum WQ-01;
the Lactobacillus hilgardii is Lactobacillus hilgardii60 TS-2.
9. Use of the method of claim 7 or 8 for reducing the loss of dry matter in sugarcane silage and/or for maintaining the stability of the organic acid content in sugarcane silage and/or for inhibiting the increase of the ammoniacal nitrogen content in sugarcane silage and/or for maintaining the aerobic stability of sugarcane silage and/or for maintaining the abundance of lactobacilli in sugarcane silage and/or for inhibiting the proliferation of aerobic spoilage bacteria in sugarcane silage and/or for increasing the nutritional value of sugarcane silage and/or for prolonging the shelf life of sugarcane silage.
10. Lactobacillus plantarum WQ-01 with the preservation number of CGMCC No. 13318;
or Lactobacillus hilgardii60TS-2 with the preservation number of CGMCC No. 19435.
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| CN111925972A (en) * | 2020-09-09 | 2020-11-13 | 天津市农业科学院 | Lactobacillus hilgardii and application thereof |
| CN112640997A (en) * | 2020-12-21 | 2021-04-13 | 沈阳化工研究院有限公司 | Method for improving aerobic stability of silage |
| CN113122483A (en) * | 2021-05-31 | 2021-07-16 | 哈尔滨工业大学 | Rapid heat-producing microbial inoculum for aerobic composting of corn straws and application of rapid heat-producing microbial inoculum |
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